Abstract Body:
Background: Hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4), a key ion channel driving pacemaker automaticity, also serves as a marker of the first heart field (FHF). Emerging evidence from cancer and stem cell research indicates that HCN ion channels regulate proliferation and differentiation in non-excitable cells, underscoring their broader significance. However, the specific function of HCN4 in FHF progenitors remains unclear.
Hypothesis: We hypothesize that HCN4 supports the expansion of FHF progenitors and contributes to early left ventricular development by modulating proliferation and differentiation.
Methods: To model FHF differentiation in vitro, human iPSCs were directed toward ventricular cardiomyocytes (vCM). Removal of WNT inhibition on day (d) 5 marked the FHF progenitor stage. Small interfering RNA (siRNA) knockdown of HCN4 was performed at this stage to assess its role.
Results: By d8, a 72-hour siRNA knockdown of HCN4 resulted in a significant downregulation of its expression level, decreasing from 11.2 ± 0.3 in the control to 4.3 ± 0.1 (p<0.001, n=3), indicating a 51.3% knockdown efficiency. Despite this, FHF or left ventricular marker expression (TBX5, HAND1, IRX4, CTNT) remained unchanged, except for NKX2-5, which was significantly upregulated upon HCN4 knockdown from 50.7 ± 0.6 in control to 60.3 ± 4.6 (p<0.001, n=3). Functionally, spontaneous beating rates remained unchanged between the HCN4 siRNA-treated group and control (50.0 ± 0.7 vs. 48.3 ± 2.7 bpm, n=3, p>0.05) indicating that HCN4 knockdown at d5 does not significantly affect automaticity of the FHF-derived vCM at d8. Western blot analysis further revealed that HCN4 protein was absent until d7, when progenitors have reached their specification into immature vCM.
Conclusion: HCN4 is transcribed during FHF progenitor stages but is not detected at the protein level until d7. Its knockdown selectively upregulates NKX2-5 without broadly affecting other FHF markers, indicating a potential role in progenitor cell proliferation and/or commitment. The data warrants further investigation of non-canonical roles of HCN4 as a regulator of ventricular chamber development.